Protective Effects of Ginsenosides (20R)-Rg3 on H2O2-Induced Myocardial Cell Injury by Activating Keap-1/Nrf2/HO-1 Signaling Pathway

Ginsenosides (20S)-Rg3 and (20R)-Rg3 are famous rare ginsenosides from red ginseng, and their configurations in C-20 are different. This study aimed to investigate the protective mechanism of ginsenosides (20S)-Rg3 and (20R)-Rg3 on H₂O₂-induced H9C2 cells and compare their activity. The results showed that the ginsenosides (20S)-Rg3 and (20R)-Rg3 could increase the cell activity and the levels of GSH-Px, SOD and CAT, and decrease activities of LDH, MDA and ROS. Further studies showed that ginsenosides (20S)-Rg3 and (20R)-Rg3 could prevent oxidative stress injury of H9C2 cells by H₂O₂ through the Keap-1/Nrf2/HO-1 pathway. But the ML385 counteracts these effects. Interestingly, among these results, ginsenoside (20R)-Rg3 was superior to (20S)-Rg3, indicating that ginsenoside (20R)-Rg3 have a stronger effect of antioxidative stress. This study reflected that ginsenoside (20R)-Rg3 could be used as a potential Nrf2 activator and a safe effective Chinese herbal monomer in the treatment of cardiovascular disease.     

紫檀芪调节Keap-1/Nrf2/HO-1信号通路对非酒精性脂肪肝大鼠氧化应激和细胞凋亡的影响

摘要 目的：研究紫檀芪调节Kelch样ECH关联蛋白1（Keap-1）/核因子E2相关因子2（Nrf2）/血红素加氧酶-1（HO-1）信号通路对非酒精性脂肪肝（NAFLD）大鼠氧化应激和细胞凋亡的影响。方法：将60只SD大鼠随机分为对照组、模型组、紫檀芪低剂量组（30 mg/kg）、紫檀芪高剂量组（60 mg/kg）、紫檀芪（60 mg/kg）+N-（4-（2,3-二氢-1-（2''-甲基苯甲酰）-1H-吲哚-5-基）-5-甲基-2-噻唑基）-1,3-苯并二氧唑-5-乙酰胺（ML385）（30 mg/kg）组,每组12只。模型组与药物干预组大鼠以高脂饲料饲养诱导NAFLD模型,对照组大鼠以普通饲料饲养,各组连续喂养12周。以紫檀芪和ML385分组处理14 d后（对照组以等剂量生理盐水处理）,检测各组大鼠脂代谢指标[三酰甘油（TG）、总胆固醇（TC）及游离脂肪酸（FFA）水平]、肝指数、肝功能指标[谷丙转氨酶（ALT）及谷草转氨酶（AST）]水平、血清白细胞介素（IL）-17、IL-6、IL-10、氧化应激指标[丙二醛（MDA）、超氧化物歧化酶（SOD）及过氧化氢酶（CAT）]水平;原位末端标记法（TUNEL）染色检测各组大鼠肝细胞凋亡率;蛋白免疫印迹法检测各组大鼠肝组织凋亡相关蛋白及Keap-1/Nrf2/HO-1通路相关蛋白表达。结果：与对照组相比,模型组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平显著降低（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平显著升高（P＜0.05）。与模型组相比,紫檀芪低、高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平均升高（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1、Bax表达水平均降低（P＜0.05）;与紫檀芪低剂量组相比,紫檀芪高剂量组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平升高（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Keap-1及Bax表达水平降低（P＜0.05）;与紫檀芪高剂量组相比,紫檀芪+ML385组大鼠血清IL-10、SOD及CAT水平、肝组织Nrf2、HO-1、Bcl-2表达水平降低（P＜0.05）,TG、TC及FFA水平、肝指数、ALT及AST水平、血清IL-17、IL-6、MDA水平、肝细胞凋亡率、肝组织Bax表达水平升高（P＜0.05）。结论：紫檀芪可能通过激活Keap-1/Nrf2/HO-1信号通路,改善NAFLD大鼠脂代谢水平,调节炎症反应及氧化应激,减轻肝组织脂肪变性及细胞凋亡。     

氧化和化学应激的防御性转导通路——Nrf2/ARE

Nrf2/ARE是近年新发现的机体抵抗内外界氧化和化学等刺激的防御性转导通路．生理条件下,NF-E2相关因子2(Nrf2,NF-E2-related factor 2)在细胞质中与Keap1结合处于非活性、易降解的状态.在内外界自由基和化学物质刺激时,Keap1的构象改变或者Nrf2直接被磷酸化,导致Nrf2与Keap1解离而活化．活化的Nrf2进入细胞核,与抗氧化反应元件(ARE)结合,启动ARE下游的Ⅱ相解毒酶、抗氧化蛋白、蛋白酶体/分子伴侣等基因转录和表达以抵抗内外界的有害刺激．MAPK、PI3K/AKT、PKC等信号通路分子广泛参与了Nrf2的活化和核转位过程,但是具体何种通路被激动、何种通路发挥主导作用,取决于刺激物种类、刺激方式和细胞类型．本文就Nrf2分子结构、Nrf2活化机制、Nrf2/ARE调控的下游基因、与Nrf2相关的信号通路分子以及其在肿瘤、炎症、衰老等应用领域的最新进展进行综述．     

Nrf2/HO-1信号通路在人诱导性多能干细胞氧化应激中的作用

氧化应激是诱导性多能干细胞(induced pluripotent stem cell, iPSC)在培养和应用中遇到的一个关键问题,探讨其作用机制具有重要的理论和实践意义。目前有关iPSC氧化应激的研究相对较少,Nrf2/HO-1信号通路在其中的作用尚不明了。因此,本研究以不同浓度的H2O2(100、200、300、400 μmol/L)处理人iPSC(hiPSC),分别在4 h和24 h于倒置显微镜下观察hiPSC及其饲养层细胞SNL氧化损伤的程度,通过碱性磷酸酶（alkaline phosphatase, AP）试剂盒和超氧化物阴离子荧光探针,分别检测hiPSC多能性和细胞活性氧(reactive oxygen species, ROS)水平,并通过qRT-PCR检测H2O2处理4 h后早期应激状态下Nrf2和HO 1 mRNA的表达水平,免疫细胞化学和Western印迹检测p-Nrf2和HO-1蛋白质的表达量。结果表明：hiPSC和SNL细胞的ROS水平呈H2O2剂量依赖性升高。除了100 μmol/L H2O2组hiPSC的细胞形态和多能性保持较好外,其余浓度H2O2均导致hiPSC出现不同程度损伤和死亡。但与SNL细胞相比,hiPSC中ROS水平相对较低,细胞状态也相对较好。SNL细胞中Nrf2和HO-1-mRNA表达的变化幅度与H2O2浓度呈线性相关,而hiPSC中Nrf2和HO-1表达的变化幅度与H2O2浓度之间并未呈现线性相关,其中Nrf2在100 μmol/L H2O2组表达量最高,而HO-1在200 μmol/L H2O2组表达量最高,意味着hiPSC氧化应激调控机制的复杂性。综上结果表明,hiPSC具有较好的抗氧化能力,其相关机制与Nrf2/HO-1信号通路有关,同时也可能涉及到其它相关通路的交互作用。     

Gain-of-Function Mutations in RIT1 Cause Noonan Syndrome,a RAS/MAPK Pathway Syndrome

RAS GTPases mediate a wide variety of cellular functions, including cell proliferation, survival, and differentiation. Recent studies have revealed that germline mutations and mosaicism for classical RAS mutations, including those in HRAS, KRAS, and NRAS, cause a wide spectrum of genetic disorders. These include Noonan syndrome and related disorders (RAS/mitogen-activated protein kinase [RAS/MAPK] pathway syndromes, or RASopathies), nevus sebaceous, and Schimmelpenning syndrome. In the present study, we identified a total of nine missense, nonsynonymous mutations in RIT1, encoding a member of the RAS subfamily, in 17 of 180 individuals (9%) with Noonan syndrome or a related condition but with no detectable mutations in known Noonan-related genes. Clinical manifestations in the RIT1-mutation-positive individuals are consistent with those of Noonan syndrome, which is characterized by distinctive facial features, short stature, and congenital heart defects. Seventy percent of mutation-positive individuals presented with hypertrophic cardiomyopathy; this frequency is high relative to the overall 20% incidence in individuals with Noonan syndrome. Luciferase assays in NIH 3T3 cells showed that five RIT1 alterations identified in children with Noonan syndrome enhanced ELK1 transactivation. The introduction of mRNAs of mutant RIT1 into 1-cell-stage zebrafish embryos was found to result in a significant increase of embryos with craniofacial abnormalities, incomplete looping, a hypoplastic chamber in the heart, and an elongated yolk sac. These results demonstrate that gain-of-function mutations in RIT1 cause Noonan syndrome and show a similar biological effect to mutations in other RASopathy-related genes.     

Marburgvirus Hijacks Nrf2-Dependent Pathway by Targeting Nrf2-Negative Regulator Keap1

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Deletion of Snf1 Affects the Nutrient Response of Yeast and Resembles Mutations Which Activate the Adenylate Cyclase Pathway

We have isolated a snf1/ccr1 mutant of Saccharomyces cerevisiae which loses viability upon starvation and fails to accumulate glycogen in response to abrupt depletion of phosphate or glucose. A snf1 null mutant is sensitive to heat stress and starvation and fails to accumulate glycogen during growth in rich medium. The phenotypes of the snf1 mutants are those commonly associated with an overactivation of the adenylate cyclase pathway. Mutations in adenylate cyclase or RAS2 which decrease the level of cAMP in the cell moderate the snf1 phenotype. In contrast, a mutation in RAS2 (RAS2val19) which increases the level of cAMP or a mutation in the regulatory subunit (BCY1) of cAMP-dependent protein kinase which results in unregulated cAMP-dependent protein kinase activity accentuates the snf1 phenotype. However, the action of SNF1 in the stress response appears at least partly independent of cAMP-dependent protein kinase because a snf1 phenotype is observed in a strain that lacks all three of the genes that encode the catalytic subunits of cAMP-dependent protein kinase. SNF1 therefore acts at least in part through a cAMP-independent pathway.     

Penehyclidine Hydrochloride Pretreatment Ameliorates Rhabdomyolysis-Induced AKI by Activating the Nrf2/HO-1 Pathway and Allevi-ating Endoplasmic Reticulum Stress in Rats

Acute kidney injury (AKI) is one of the most severe complications of rhabdomyolysis (RM). The underlying mechanisms and potential preventions need to be investigated. Penehyclidine hydrochloride (PHC) was reported to ameliorate renal ischemia-reperfusion injury, but the effect of PHC on RM-reduced AKI is unknown. In this study, we established a rat model of RM-induced AKI using an intramuscular glycerol injection in the hind limbs. Rats were pretreated with PHC before the glycerol injection, and the heme oxygenase-1 (HO-1) inhibitor ZnPP was introduced to evaluate the effect of HO-1 on RM-induced AKI. PHC pretreatment ameliorated the pathological renal injury and renal dysfunction, and decreased the renal apoptosis rate in RM-induced AKI. PHC significantly up-regulated HO-1 expression, increased HO-1 enzymatic activity and decreased the accumulation of myoglobin in renal tissues. This effect was partly inhibited by ZnPP. PHC pretreatment also effectively up-regulated nuclear factor erythroid 2-related factor 2 (Nrf2) and down-regulated glucose regulated protein 78 (GRP78) and caspase-12 at both the gene and protein levels. These results suggest that the protective effects of PHC pretreatment on RM-induced AKI occur at least in part through activating the Nrf2/HO-1 pathway and alleviating endoplasmic reticulum stress (ERS) in rat renal tissues.     

DHTKD1 Mutations Cause 2-Aminoadipic and 2-Oxoadipic Aciduria

Abnormalities in metabolite profiles are valuable indicators of underlying pathologic conditions at the molecular level. However, their interpretation relies on detailed knowledge of the pathways, enzymes, and genes involved. Identification and characterization of their physiological function are therefore crucial for our understanding of human disease: they can provide guidance for therapeutic intervention and help us to identify suitable biomarkers for monitoring associated disorders. We studied two individuals with 2-aminoadipic and 2-oxoadipic aciduria, a metabolic condition that is still unresolved at the molecular level. This disorder has been associated with varying neurological symptoms. Exome sequencing of a single affected individual revealed compound heterozygosity for an initiating methionine mutation (c.1A>G) and a missense mutation (c.2185G>A [p.Gly729Arg]) in DHTKD1. This gene codes for dehydrogenase E1 and transketolase domain-containing protein 1, which is part of a 2-oxoglutarate-dehydrogenase-complex-like protein. Sequence analysis of a second individual identified the same missense mutation together with a nonsense mutation (c.1228C>T [p.Arg410^∗]) in DHTKD1. Increased levels of 2-oxoadipate in individual-derived fibroblasts normalized upon lentiviral expression of the wild-type DHTKD1 mRNA. Moreover, investigation of L-lysine metabolism showed an accumulation of deuterium-labeled 2-oxoadipate only in noncomplemented cells, demonstrating that DHTKD1 codes for the enzyme mediating the last unresolved step in the L-lysine-degradation pathway. All together, our results establish mutations in DHTKD1 as a cause of human 2-aminoadipic and 2-oxoadipic aciduria via impaired turnover of decarboxylation 2-oxoadipate to glutaryl-CoA.     

Garlic Attenuates Cardiac Oxidative Stress via Activation of PI3K/AKT/Nrf2-Keap1 Pathway in Fructose-Fed Diabetic Rat

Background

Cardiovascular complication due to diabetes has remained a major cause of death. There is an urgent need to intervene the cardiac complications in diabetes by nutritional or pharmacological agents. Thus the present study was designed to find out the effectiveness of garlic on cardiac complications in insulin-resistant diabetic rats.

Methods and Results

SD rats were fed high fructose (65%) diet alone or along with raw garlic homogenate (250 mg/kg/day) or nutrient-matched (65% corn starch) control diet for 8 weeks. Fructose-fed diabetic rats showed cardiac hypertrophy, increased NFkB activity and increased oxidative stress. Administration of garlic significantly decreased (p<0.05) cardiac hypertrophy, NFkB activity and oxidative stress. Although we did not observe any changes in myocardial catalase, GSH and GPx in diabetic heart, garlic administration showed significant (p<0.05) increase in all three antioxidant/enzymes levels. Increased endogenous antioxidant enzymes and gene expression in garlic treated diabetic heart are associated with higher protein expression of Nrf2. Increased myocardial H₂S levels, activation of PI3K/Akt pathway and decreased Keap levels in fructose-fed heart after garlic administration might be responsible for higher Nrf2 levels.

Conclusion

Our study demonstrates that raw garlic homogenate is effective in reducing cardiac hypertrophy and fructose-induced myocardial oxidative stress through PI3K/AKT/Nrf2-Keap1 dependent pathway.     

MicroRNA-93 Downregulation Ameliorates Cerebral Ischemic Injury Through the Nrf2/HO-1 Defense Pathway

The present study was designed to evaluate the potential role of miR-93 in cerebral ischemic/reperfusion (I/R) injury in mice. The stroke model was produced in C57BL/6 J mice via middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion. And miR-93 antagomir was transfected to down-regulate the miR-93 level. Our results showed that miR-93 levels in the cerebral cortex of mice increased at 24 and 48 h after reperfusion. Importantly, in vivo study demonstrated that treatment with miR-93 antagomir reduced cerebral infarction volume, neural apoptosis and restored the neurological scores. In vitro study demonstrated that miR-93 antagomir attenuated hydrogen peroxide (H₂O₂)-induced injury. Moreover, miR-93 antagomir suppressed oxidative stress in I/R brain and H₂O₂ treated cortical neurons. Furthermore, we founded that down-regulation of miR-93 increased the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1) and the luciferase reporter assay confirmed that miR-93 directly binds to the predicted 3′-UTR target sites of the nrf2 gene. Finally, we found that knockdown of Nrf2 or HO-1 abolished miR-93 antagomir-induced neuroprotection against oxidative stress in H₂O₂ treated neuronal cultures. These results suggested that miR-93 antagomir alleviates ischemic injury through the Nrf2/HO-1 antioxidant pathway.     

丹参酮治疗冠心病对Nrf2/ARE信号通路和氧化应激的影响

摘要 目的：分析丹参酮通过核因子E2相关因子2（Nrf2）/抗氧化反应元件（ARE）信号通路发挥抑制冠心病患者的氧化应激和心肌损伤的作用。方法：前瞻性纳入2022年6月至2023年6月我院诊断不稳定心绞痛患者84例为研究对象,随机分为对照组和观察组,每组各42例,对照组采用常规药物和介入干预,观察组加用丹参酮ⅡA磺酸钠注射液（剂量40 mg/d）,连续治疗2周。比较两组治疗前后外周血Nrf2、ARE、缺氧诱导因子-1α（HIF-1α）和Caspase-1蛋白表达量,氧化应激指标活性氧簇（ROS）、丙二醛（MDA）和超氧化物歧化酶（SOD）,炎症介质肿瘤坏死因子（TNF）-α和白细胞介素（IL）-1β,心肌损伤指标肌钙蛋白I（cTnI）和肌酸激酶同工酶（CK-MB）。结果：两组治疗前上述指标均无明显差异（P＞0.05）,治疗后与治疗前比较改善明显（P＜0.05）,且观察组Nrf2和ARE蛋白表达量显著高于对照组,而HIF-1α和Caspase-1蛋白表达量显著低于对照组,ROS水平下降,MDA和SOD水平升高,TNF-α和IL-1β降低,cTnI和CK-MB水平下降（P＜0.05）。结论：丹参酮可能通过激活Nrf2/ ARE信号通路发挥抑制冠心病患者的氧化应激、炎症反应、细胞凋亡以及心肌损伤的作用。